Detection of inflammation

ABSTRACT

The invention relates to a method for the detection of inflammations, in particular of inflammatory processes in the tissue of mammary glands in humans or animals, in particular in cattle and a test-kit for carrying out said method. The method is particularly suitable for the specific detection of inflammations in mammary glands, for example during testing of milk, whereby the enzyme implicated in inflammatory processes prostaglandin D synthase (PGDS) is qualitatively or quantitatively determined.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/EP02/03383, filed Mar. 26, 2002, and designating the U.S.

DESCRIPTION

The present invention relates to a method for detection of inflammationprocesses. The present invention relates in particular to a method fordetection of inflammation processes in tissue in the mammary glands ofhumans or animals, in particular cattle, and to a test kit for carryingout the method according to the invention. The method is particularlysuitable for specific detection of inflammation in mammary glands, forexample by examining milk, with the enzyme Prostaglandin D Synthase(PGDS) which is involved in the occurrence of inflammation beingdetermined qualitatively or quantitatively.

Inflammation in cattle mammary glands is of major financial importancebecause of the fact that it arises frequently in about 30-40% of allmilk cows. On the one hand, inflammation such as this reduces the amountof milk produced and the quality of the milk for marketing. On the otherhand, excessively late identification and the massive antibiotictreatment that is then required result in waiting times in which themilk cannot be marketed. Assuming that 10% of the milk yield from 15% ofthe milk cows in the Federal Republic of Germany is lost, this isequivalent to an average production of 6500 kg of milk, and with a priceper kilogram of DM 0.45, this can be calculated to result in a financialloss of at least about DM 210 million per annum.

All diagnostic methods used so far for detection of mammary glandinflammation are non-specific and do not allow direct assessment of theinflammation processes in the mammary gland. These known methodsinclude, for example, measurement of the electrical conductivity of themilk, determination of the cell counts or detection of enzymes whichindicate that tissue damage has already occurred, such aslactatdehydrogenase. However, enzymes such as these cannot be detecteduntil relatively late.

Currently available fast tests for on sight use are indirect tests andare based on qualitative and/or semi-quantitative detection ofdeoxyribonucleic acid which are released from cells contained in themilk and are assessed visually. An increased number of cells, whichresults in an increase in the quantity of deoxyribonucleic acid, is inthis case regarded as an indicator of inflammation in the mammaryglands. While the test gives an indication of the number of cells, anincreased number of cells is, however, not caused directly byinflammation processes, as can be seen, for example, from the highpositive result which can be observed at the start and at the end oflactation.

Since inflammation of the mammary glands is caused virtually exclusivelyby bacterial infections, bacteriological diagnosis should be regarded asa major factor. However, in practice, it has been found that there israrely any correlation between the stated non-specific changes in themilk and bacteriology. Thus, until now, diagnosis in particular ofsubclinical inflammation has been highly uncertain. Examples offunctional protein indicators for subclinical mastitis are, for example,LDH, NAGase (Zank W., Schlatterer B.

Assessment of subacute mammary inflammation by soluble biomarkers incomparison to somatic cell counts in quarter milk samples from dairycows. J Vet Med A 1998; 45:45-51), plasminogen and plasmin (Urech E.,Puhan Z., Schällibaum M. Changes in milk protein fraction as affected bysubclinical mastitis. J Dairy Sci 1999; 82:945-951) as well as themarker C5a (Rainard P., Poutrel B. Generation of complement fragment C5ain milk is variable among cows. J Dairy Sci 1999; 82:2402-2411).

Inflammation is generally triggered by inflammation mediators afterinitiation by bacterial metabolism products. Initial processes are inthis case based on a locally increased phospholipid metabolism, whichleads to prostaglandins and to metabolism products similar toprostaglandin. Although these can be detected in the milk secretion froman inflamed mammary gland, no substance patterns which an be assesseduniquely have, however, yet been detected or identified, and this isrelated inter alia to the complexity of and the effort for analysis (F.Atroshi et al. Inflammation-related changes in cyclic AMP and cyclic GMPin bovine mastitis. Vet Res Commun 1989; 13:427-33; O. O'Sullivan et al.Analysis of prostaglandin D2 metabolites in urine: comparison betweenenzyme immunoassay and negative ion chemical ionisation gaschromatography-mass spectrometry. Prostaglandins Other Lipid Mediat1999; 57:149-165).

One object of the invention was therefore to provide a method and, inparticular, a specific inflammation indicator for detection of mammarygland inflammation. It would also be desirable to be able to detectother inflammation processes with the aid of an inflammation indicator.A further object of the invention was therefore to provide a method fordetection of inflammation.

This object is achieved by a method for detection of inflammation whichis characterized in that the enzyme prostaglandin D synthase (PGDS) orparts of it is or are determined qualitatively and/or quantitatively.The determination is preferably made in bodily fluids. It has been foundthat the prostaglandin D synthase (PGDS) rises during inflammationprocesses in the body and in the tissue of humans and animals. In thecase of inflammation, for example in the case of rheumatoid arthritis orencephalitis, increased amounts of PGDS can be found in bodily fluids,for example in the plasma, in the synovial fluid, in the cerebrospinalfluid, in the urine or in the ejaculate. Prostaglandin D synthase (PGDS)is thus suitable for use as an inflammation indicator for inflammationprocesses, since the amount of the enzyme rises when inflammation ispresent. The determination can also be made in the stool, and is thenused to detect inflammation in the gastrointestinal tract, in particularin the stomach, pancreas or intestines.

The invention relates in particular to a method for detection of mammarygland inflammation, which is characterized in that the enzymeprostaglandin D synthase (PGDS) or parts of it is or are determinedqualitatively and/or quantitatively.

It has been found that the prostaglandin D synthase (PGDS) isprecipitated to an increased extent in the case of mammary glandinflammation, so that this enzyme is suitable for use as an inflammationindicator for specific qualitative and/or quantitative detection ofmammary gland inflammation.

Thus, according to the invention, an indicator with high specificity andwhich is coupled directly to the inflammation processes is provided,whose detection and quantification also allow early diagnosis ofinflammation, in particular inflammation of the mammary glands. Theindicator enzyme prostaglandin D synthase (access number 002853) occursin a number of isoforms, which can be detected individually or together.

The indicator substance PGDS for detection of mammary gland inflammationwas found by comparison of the whey protein pattern of milk from animalsaffected by mammary gland inflammation with that from unaffected,healthy animals. This protein pattern can be obtained, for example, bytwo-dimensional gel electrophoresis (2D-PAGE), with four additionalspots with a molecular weight of about 26 kDa and a pH range from 5.0 to6.4 occurring in milk samples from animals with mammary glandinflammation. The substances removed from the gel showed a very similarchromatographic and mass-spectrometric pattern of chemotryptic peptides.A databank search revealed the four substances as isoforms of cattleprostaglandin D synthase (PGDS) and, in one of the isoforms, a cysteinresidue was oxidized to form a sulfonic acid.

Despite the highly complex nature of the defensive and inflammationmechanisms in mammary glands against the invasion of various infectiousagents, it was thus possible to produce a marker which reliably allowsqualitative and/or quantitative detection of mammary gland inflammation.On the basis of the large quantity of caseins in the milk,prefractionation of cattle milk proteins was carried out with respect towhey components, as a result of which it was possible to achieve apolypeptide resolution which allowed the identification of the PGDSmarker enzyme according to the invention.

Surprisingly, this marker molecule could be detected not only in theblood or within the cells in the event of mammary gland inflammation,but also occurs to an increased extent in the milk in the case ofmammary gland inflammation, so that easy and simple detection of mammarygland inflammation is possible. Furthermore, the enzyme could bedetected to an increased extent in bodily fluids when inflammation waspresent, for example to an increased extent in the cerebrospinal fluidin the case of encephalitis, and in the synovial fluid in the case ofrheumatoid arthritis.

Prostaglandin D synthase, which is similar to lipocalin and is alsoreferred to as prostaglandin-H2-D-isomerase (EC5.3.99.2), is amembrane-bound enzyme (Giacomelli S., Leone M. G., Grima J. SilvestriniB., Cheng C. Y. Astrocytes synthesize and secrete prostaglandin Dsynthase in vitro. Biochim Biophys Acta 1996; 1310:269-276).

In principle, the method according to the invention can be used fordetection of inflammation in humans or animals.

While the method according to the invention can preferably be used fordetection of mammary gland inflammation in humans or animals, it isparticularly preferably used for detection of mammary gland inflammationin cattle. The enzyme prostaglandin D synthase or parts of it arepreferably determined, particularly parts with a length of at least 15amino acids, and more preferably of at least 30 amino acids, and evenmore preferably of at least 50 amino acids, in a bodily fluid sample orpreferably in a milk sample. Human prostaglandin D synthase is used inparticular for detection in humans, and bovine prostaglandin D synthaseis used in particular for detection in cattle. The determination of themarker in a milk sample allows quick and simple assessment on sight asto whether or not inflammation is present, and as to the severity of anyinflammation.

The determination is particularly preferably made using at least oneantibody to prostaglandin D synthase. Antibodies which can be usedaccording to the invention may be produced using methods known from theprior art. Those skilled in the art will be highly familiar with methodsfor producing monoclonal and polyclonal antibodies. The antigen, that isto say in the present case the enzyme prostaglandin D synthase or partsof it, is or are used by way of example to produce monoclonalantibodies, usually purified in order to produce antibodies. Inprinciple, the method which was first described by Köhler and Millsteincan be used for this purpose, and those skilled in the art will also befamiliar with modified forms and further developments of these methods.The selectivity of the antibodies obtained can be confirmed byselection.

On the basis of prostaglandin D synthase or parts of it, it is alsopossible to produce polyclonal antibodies using known methods.Polyclonal antibodies such as these are preferably used, in particularpolyclonal antibodies which identify all isoforms of prostaglandin. Dsynthase.

The antibody is particularly preferably produced using the peptidesPGDS1 (Ac-LTSTFLRKDQCETRTLL-NH2) [SEQ ID NO:1], PGDS2(Ac-FEEDKFLGRWFTSGLAS-NH2) [SEQ ID NO:2], or PGDS3(Ac-GPGQDFRMATLYSRSQ-NH2) [SEQ ID NO 3].

The determination is preferably made by means of an immunoassay, inparticular by means of a quantitative sandwich ELISA. In one preferredembodiment of this test, a sample, for example a milk sample to beexamined, is brought into contact with at least two different receptors,the first receptor R1 of which is in an immobilized form in a solidphase and can be bonded with PGDS, while the second receptor R2 is inthe liquid phase, and can likewise be bonded with PGDS and carries amarker or allows bonding to a molecule which can be detected, separatesthe solid phase from the liquid phase, and determines the marking or thedetectable molecule in the solid phase. The amount of PGDS in the samplecan be quantified by quantifying the determined amount of the marking orof the detectable molecule.

In a further preferred embodiment, the method according to the inventionis carried out as a Western Blot.

When carrying out an immunoassay, it is possible to specificallydetermine the content of PGDS in a sample, for example in a milk sample,which can be regarded as a marker for mammary gland inflammation. Inaddition to the quantitative determination, a qualitative determinationis also possible in order, for example, to obtain a rapid result inadvance. A cut-off value is preferably defined, in order to distinguishbetween negative and positive results.

In one preferred embodiment, at first an antibody which specificallybinds with the enzyme PGDS is immobilized in a solid phase by means ofknown methods. The sample to be investigated, in particular a milksample is, after being skimmed, brought into contact with the fixedantibody in some suitable buffer solution, and is bonded via this to thesolid phase. After washing the immobilized antibody enzyme complexobtained in this way, a further, secondary antibody is then added, whichis coupled to a marker, which binds to a different epitope in the PGDS,and determines the amount of marker that remains in the system. Theamount of the bound marker is directly proportional to the amount ofPGDS in the sample. The method according to the invention is preferablycarried out in parallel with reference material with differentconcentrations of PGDS in bodily fluid samples, in particular in milksamples, in order to provide a high level of analysis confidence andaccuracy.

The method according to the invention makes it possible to detect and toquantify both inflammation that is about to start and already existinginflammation, particularly mammary gland inflammation. Furthermore, themethod can be carried out specifically and easily. The method accordingto the invention is furthermore suitable not only for being carried outin a laboratory, for example as a quantitative test method, but may alsobe in the form of a quick-test variant, which allows the location andseverity of inflammation to be assessed in situ.

In addition to carrying out the method according to the invention in theform of an immunoassay and, in particular, of an ELISA or a WesternBlot, PGDS can also be detected, for example in milk, with the aid ofother detection methods, with the detection being preferable inparticular with the aid of mass spectroscopy, in particular MALDI-TOF(matrix-assisted laser desorption/ionization time of flight massspectroscopy) or by means of gel electrophoresis, in particulartwo-dimensional gel electrophoresis. In addition, the analyteconcentration can in principle also be determined by means ofbiosensors, such as amperometric sensors, potentiometric, piezoelectric,thermometric or photometric sensors, or else by means of semiconductorelectrodes, such as field-effect transistors (FETs), chemosensitivefield-effect transistors (CHEMFETs), suspended gate field-effecttransistors (SGFETs) or ion-sensitive field-effect transistors.Biosensors such as these are described, for example, by E. A. H. Halland G. Hummel in “Biosensors”, Springer-Verlag Heidelberg, Germany 1995.The detection can also be carried out by means of the Kandelabertechnology from IBM, Inc. A particularly advantageous procedure withregard to sensitivity, dynamic measurement range, analysis kinetics andformat flexibility can be achieved by the use ofelectrochemoluminescence technology. Electrochemoluminescence is aprocess in which light is released. The release of light is induced byapplying an electrical potential to an electrode which mediates a cyclicredox reaction of a ruthenium metal ion (Bruno, G. (1997) Rec. Rp. pages175-179; Williams R. (1996), Amer. Biotech., page 27). A similarsuitable technology is the TRACE technology from the company CIS,Germany.

The present invention also relates to a test kit for detection and/orfor diagnosis of inflammation, in particular of mammary glandinflammation, which contains at least one receptor which binds withprostaglandin D synthase, with the test kit being designed in particularfor carrying out the method according to the invention. The test kit isintended in particular for detection in the case of cattle, but can alsobe provided for detection in the case of other animals or humans.

The antibody is preferably a polyclonal antibody which binds with anumber of the isoforms, and preferably with all of the isoforms, ofprostaglandin D synthase.

The receptor which binds with the enzyme PGDS preferably allows bindingto a solid phase, so that it is possible to separate the liquid phase,containing the sample, in particular a milk sample, and the solid phasewith the PGDS bound to it via a receptor. This solid phase is likewise apart of the test kit, in one preferred embodiment. The sample to beinvestigated is then brought into contact, in a suitable buffersolution, with the fixed or fixable receptor, in particular an antibody,and is bound to the solid phase by means of the receptor. When usingmilk as the sample, the cream is preferably removed before this step.

After washing the immobilized antibody PGDS complex that is obtained, afurther marked secondary antibody, or a secondary antibody which can bebound to a detectable molecule, for example with biotin, is then addedand preferably binds to another epitope of the PGDS. The marking canthen be determined, for example with the aid of streptavidin peroxidase.The amount of bound marker is directly proportional to the amount ofPGDS in the sample. The test kit expediently contains reference materialof a known content and, in particular, with different concentrations ofPGDS for quantitative determination and for analysis quality confidence.

In addition to the preferred form as a solid phase sandwich assay, themethod according to the invention can also be carried out using otherdetection methods. In particular, all types of immunological detectionmethods which are carried out with the aid of antibodies can be usedaccording to the invention. Thus, for example, the method according tothe invention can be carried out as a sandwich, ELISA, oscillatingcrystal, microbalance or electrochemoluminescence test, and preferablycontains the reactants required for this purpose, and possibly alsoapparatuses.

For simple handling, the test may be in the form of a test strip onwhich the required antibodies are arranged in different zones, forexample, fixed either in soluble form or in solid phases. The sample orthe liquid component of the sample or an extract of it can then migratethrough the test strip and can produce a signal at the detection pointwhen PGDS is present in the sample.

In a further preferred embodiment, the test is designed as a fast testin the form of a membrane diffusion test, for example as animmunochromatographic lateral flow test, or some similar test.

For the purposes of the present invention, the expressions “receptor”and “antibody”, respectively, also means those parts or fragments ofreceptors or antibodies which still provide the necessary binding toPGDS. In this case, it is also possible to use a conjugate, for examplecomposed of two antibodies, instead of a single antibody. In particular,for example, an antibody which can bind with PGDS can be used as thesecondary receptor, with the detection being carried out using a furthermarked antibody, which is directed against the Fc part of the secondaryantibody and carries a marking, or is once again cobbled to a detectablemolecule. Such conjugate formation from two antibodies is also intendedto be covered within the scope of the present invention, if thesecondary antibody is defined such that it allows binding to adetectable molecule. An analogous situation applies to the binding ofthe first antibody to the solid phase. This binding can also take placevia antibodies coupled to the solid phase and which bind the Fc part ofthe first antibody.

For the purposes of the present invention, it is preferable for the testkit to contain antibodies or antibody fragments which can bind in aspecific manner as receptors. It is furthermore preferable for the firstantibody to be coupled to a solid phase, while the second antibody ispreferably a soluble, marked antibody or a soluble antibody which isbounded to an enzyme which can in turn be determined via a detectionreaction. The marking may be designed such that it can be identifiedwithout any further additions of substances, for example a gold markingor a fluorescent marking, or else be designed so as to allow theaddition of further reactants to produce the determination. For example,the detection can be made by marking with enzymes and the addition ofthe enzyme substrate.

A further object of the present invention is the use of an antibodyagainst prostaglandin D synthase for producing means for detection ofinflammation, in particular of mammary gland inflammation. According tothe invention, it has been found that antibodies against prostaglandin Dsynthase are specific markers for inflammation, in particular formammary gland inflammation, so that they can be used both forqualitative and for quantitative detection of such inflammation.

A further object of the present invention is a monoclonal antibody whichspecifically binds the enzyme prostaglandin D synthase (PGDS).

The antibodies according to the invention can be obtained with the aidof methods that are known per se. In this case, the enzyme prostaglandinD synthase (PGDS) is first of all isolated and if necessary purified.After this, an experimental animal can be immunized with theprostaglandin D synthase obtained in this way, or with fractions of itwhich have the corresponding epitopes, and the antibodies which areformed can be isolated. Fractions which are used for immunization may,for example, originate from protease digestion of the purified PGDS, ormay comprise synthetic partial peptides of it. The production of suchpartial peptides is known per se to those skilled in the art. In thiscase, it is possible, for example, to use computer programs to choosefrom the overall sequence elements which contain appropriate epitopes.These sequences are then tested for their usefulness for producingspecific antibodies.

The monoclonal antibodies according to the invention are preferablyproduced using the methods from Köhler, Millstein (Nature 256, 495-497(1975)). In this case, by way of example, BALB/c mice are immunized withisolated PGDS, and the spleen cells from these animals are fused with amyeloma cell line, for example, PA I. The secreted antibodies are testedfor their specific nature and are isolated using, for example, ELISA orRIA.

The present invention also relates to aptameres which specifically bindto PGDS. Aptameres are oligonucleotide sequences which have specificbinding characteristics. The aptameres according to the invention may,for example, be produced and identified using the methods described inU.S. Pat. No. 5,270,163 or in Sumedha, Clin. Chem. 45 (1999) 1628-1650.

The invention will be explained in more detail using the followingexamples and the attached figures.

FIG. 1:

Comparison of whey protein patterns of inflamed (FIG. 1A) and unchanged(FIG. 1B) mammary gland quarters with SDS-2D-PAGE.

Equivalent amounts (6 mg) of lyophilized protein samples were loadedonto pH 4-7 linear IPG strips and were focused, followed byelectrophoresis on an SDS gel with a constant acrylamide concentration(12% T, 2.67% C). Arrows indicate the identified protein species, whichare not present in the unchanged udder quarter. The spots annotated withletters represent identified proteins which are normally found in anymilk: A: IgG1; B: beef serum albumin fragment; C: beef serum albuminfragment; D: beef serum albumin fragment; E: K-Casein; F: EPI (secretoryprotein).

FIG. 2:

The reverse phase HPLC separation of the chymotryptic peptides, obtainedby digestion of the proteins related to the spots 2 and 3 in the gel.

FIG. 3:

MALDI-TOF spectra in the negative ion mode of the chymotryptic peptides,which were obtained by digestion of the proteins in the spots 2 and 3 inthe gel. The spectra are virtually identical apart from the peptidesmarked by arrows. In the spot 3, the peptides for 1.085 and 1.198 Da canbe associated with the residues 68 to 77 and 67 to 77 in the PGD-Ssequence. The masses of the corresponding peptides in the spot 2 are 9Da less.

FIG. 4:

PGDS sequence [SEQ ID NO: 4]. The observed chymotryptic peptides areunderlined. Peptides which are different in the spots 2 and 3 are shownin bold.

FIG. 5:

Western Blots of milk secretions from differently changed udderquarters.

EXAMPLES Example 1

Materials and Methods

Seven cows were investigated, chosen on the basis of a bacteriologicalexamination, with regard to the somatic cell count (SCC) and theactivity of the lactate dehydrogenase (LDH). Initial milk samples ofinfected and uninfected quarters of the same udder were placed directlyin 50 ml containers, which contained a combination of the two proteinaseinhibitors PMSF (phenylmethylsulfonyl fluoride, 2 nM) and APMSF(2-(4-amidinophenyl)methanesulfonyl fluoridehydrochloride, 20 μm), bothfrom Roche Diagnostics Mannheim. Non-spiked initial milk samples wereused for determining the cell counts and the LDH activity.

1.1 Analysis and Production of Whey Fractions

SSC was measured using a Fossomatic 360 (Foss Electric, Hillerfod,Denmark). The LDH activity in skimmed milk was measured using a BeckmanSynchron CX5 CE auto analyzer (Zank W, Schlatterer B. Assessment ofsubacute mammary inflammation by soluble biomarkers in comparison tosomatic cell counts in quarter milk samples from dairy cows. J. Vet.Med. A 1998; 454:45-51). The results were expressed in LDH units(μmol/sec 1). The whey protein fractions were produced using the methodby Molloy M P, Herbert B R, Yan J X, Williams K L, Gooley A A.(Identification of wallaby milk proteins separated by two-dimensionalelectrophoresis, using amino acid analysis and sequence tagging.Electrophoresis 1997; 18:1073-1078). In short, milk fat was separated bycentrifuging 5000 g at 4° C. for 10 minutes. Casein was precipitated byincreasing the acidity of skimmed milk to a pH of 4.6 using 4 M aceticacid, and was removed by centrifuging 5000 g at 4° C. and by filtration,initially by means of a paper filter and then by means of apolyvinylidene difluoride membrane (PVDF, 0.2 μm). The filtrate wasdialyzed over night at 4° C. against double-distilled water. Undissolvedmaterial was removed by centrifuging. A protein determination was madebased on Bradford M H. (A rapid and sensitive method for quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 1976; 205:22-26) using a protein assay fromBio-Rad (Hercules, USA). Aliquots was lyophilized and stored at −70° C.

1.2 Two-dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

1.2.1 First Dimension: Isoelectric Focusing

6 mg of lyophilized proteins from the whey fractions were dissolved in350 μl of rehydration solution (8 M urea, 2% CHAPS, 0.3%dithioerythritol (DDT), 2% IPG puffer). Immobiline™ Dry Strips, pH 4-7,18 cm, were used on Multiphor II apparatus (Amersham Pharmacia Biotech,Uppsala, Sweden). The focusing was started at 500 V. The voltage wasincreased in steps to 3500 V within one hour, and the focusing wascontinued at 2 mA and 10 W for 7.5 hours.

1.2.2 Second Dimension: SDS Polyacrylamide Gel Electrophoresis

Before the second dimension, IPG strips were first of all converted toan equilibrium form in 10 ml of 50 mM Tris-HCl, pH 8.8, 6 M urea, 30%(vol/vol) glycerin, 2% (weight/volume) SDS, a trace bromophenol blue and65 mM dithioerythritol (DDT) for 15 minutes, and were then placed in thesame buffer for a further 15 minutes, with DTT being replaced by 260 mMof iodoacetamide. The IPG strips in an equilibrium form were embedded in0.5% agarose on the cathode side on an SDS gel with a constantacrylamide concentration (12% T, 2.67% C, 200×205×1.5 mm), using thehorizontal Protean® II-apparatus (Bio-Rad, Richmond, USA). Theelectrophoresis conditions were 150 V, 15 mA/gel, 10 W at 12° C. overnight, until the bromophenol detection dye appeared at the anode end.The gels were dyed using Coomassie blue R 350 until the required dyeintensity was obtained. The color was removed from the gels byrepeatedly changing a dye removal solution (25% ethanol, 8% acidicacid).

1.3 Identification and Analysis of the Gel-dissolved Protein

1.3.1 Proteolytic Digestion of the Proteins in the Gel and Extraction ofthe Peptides

The dyed protein spots were cut out and the dye was removed over 2-6hours in a mixture of 40% (vol/vol) acetronitrile and 60% (vol/vol) 50mM NH₄HCO₃. After drying by vacuum centrifuging, they were impregnatedwith a chymotrypsin solution (30 ng/μl in 50 mM NH₄HCO₃). The digestionprocess was carried out over night at room temperature. The peptideswere extracted by the addition of 30 μl 0.1% trifluoroacetic acid (TFA)followed by 50 μl of acetonitrile (CH₃CN), after 10 minutes. The residuewas removed and the procedure was repeated once. The combined residueswere lyophilized.

1.3.2 RP-HPLC (Reverse Phase High-pressure Liquid Chromatography)Separation of the Chymotryptic Peptides

The peptides formed by enzyme digestion (chymotrypsin) were separated bymeans of RP-HPLC (SMART system, Pharmacia, Uppsala, Sweden) on aPharmacia C2/C18 SC2.1/10 column using a linear (0-50% in 40 min)acetonitrile gradient in 0.1% TFA. The peptides were detected at awavelength of 214 nanometers.

1.3.3 MALDI-TOF Mass Spectroscopy

MALDI-TOF (matrix-assisted laser desorption/ionization time of flightmass spectroscopy) mass spectra were produced on a Reflex II MALDI-TOFinstrument (Bruker-Daltonik, Bremen, Germany). All the spectra wererecorded in the reflector mode using α-cyano-4 hydroxycinnamic acid (15mg/ml in 70% acetronitrile) as a matrix. For mass-spectrometricanalysis, the peptide mixture was dissolved in 10 μl of a ˜3/7 mixture(vol/vol) of acetonitrile and 2% aqueous TFA. Individual peptides,purified by means of RP-HPLC, were subjected to a further analysis byrecording the post-source decay spectra.

1.4 Data Bank Search

The data bank search using the proteolytic peptide masses was carriedout using the Peptide Search Program developed by the EMBL Protein &Peptide Group (http://www.mann.embl-heidelberg.de orhttp://www.peptsearch.protana.com). Peptide Search uses a non-redundantprotein databank which at the moment contains more than 465,000 entries.The identification of the proteins is based on the comparison of the setof peptide masses, which were derived experimentally from the isolatedproteins, with the theoretical masses, derived on an enzyme basis, ofthe peptides, derived on an enzyme basis, for all the protein sequencesin the databank.

2. Results

2.1 Inflammation Marker

Selection criteria for subclinically inflamed mammary gland quarterswere LDH activities of more than 2 μmol/sec I, somatic cell counts ofmore than 250,000 ml⁻¹ and, where determined, positive bacteriologicalresults. Only changed quarters and unchanged quarters of the same udderwere used for comparison purposes. The udder quarters of the selectedcows which were examined are shown in Table 1.

TABLE 1 Individual cows with inflamed (1) and unchanged (2) mammarygland quarters LDH-UNITS COW QUARTER μmol/sec 1 SCC MICROBIOLOGY 1 112.23 9 911 000 not determined 2 1.21   30 000 2 1 19.19 5 784 000 notdetermined 2 4.22   203 000 3 1 16.50 5 010 000 not determined 2 1.67  29 000 4 1 2.53   984 000 streptococcus spp. 2 1.06   29 000 5 1 17.42 4048 000 staphylococcus spp. 2 1.58   162 000 6 1 28.11 7 697 000streptococcus spp. 2 2.86   50 000 7 1 5.21 1 436 000 staphylococcusspp. 2 0.99   71 0002.2 SDS-2D-PAGE Visualization of the Whey Fractions

The clearest difference in the 2D-PAGE patterns is the appearance offour spots in the whey fractions, which were obtained from inflamedquarters, compared with those from the respective internal controls. Fora molecular weight of 26 kDa, these spots are arranged over a pI rangefrom 5.2 to 6.2, and are marked with the numbers 1-4 in FIG. 1.

2.3 Identification of the Proteins Within the Spots

The spots 1-4 which were cut out of the 2D gels resulted in very similarchromatographic and mass-spectrometric patterns of chymotryptic peptides(FIGS. 2 and 3 show the patterns obtained for the spots 2 and 3). Adatabank search based on the peptide masses that were found allowed theproteins to be identified without any doubt as lipocalin-likeprostaglandin D synthase (access number O02853). The mass-spectrometricdata also made it possible to confirm that the ripe protein starts atresidue 39 in the databank sequence.

When the peptide patterns obtained from the spots 2 and 3 are compared,two differences can be detected, as indicated by the arrows in FIG. 2and FIG. 3. The corresponding peptides for the spot 3 can be associatedwith the sequence sections LLRPAGPPGCY [SEQ ID NO: 5] and LRPAGPPGCY[SEQ ID NO: 6] (see FIG. 4). The corresponding peptides for the spot 2show a mass difference of −9 Da. Post-source decay spectra for the twopeptides isolated by means of RP-HPLC made it possible to associate themodification with a cystein residue in position 76 of the ripe protein.The mass difference of −9 Da. corresponds to the difference between acystein alkylated using an iodoacetamide and a cystein oxidized to formsulfonic acid. This also matches the observation that the isoelectricpoint of the modified protein is shifted toward an acidic value. Themodification by iodoacetamide (spot no. 3) occurs during the gelelectrophoresis conditions in the second equilibration step while, incontrast, the oxidized cystein (spot 2) occurs even before the 2D gelelectrophoresis as a sulfonic acid derivative. Since the introduction ofa sulfonic acid group has a major influence on the isoelectric point ofa protein, the various oxidized cysteins may explain the observation ofa number of gel spots with very similar molecular weights but with adifferent pl. The different spots may, however, also be caused by apost-translational modification, such as a heterogeneous glycosylationpattern.

Example 2

Western Blot

Western Blots were produced from milk secretions from differentlychanged udder quarters. The polyclonal antibodies used for the Blot wereproduced against prostaglandin D synthase from bovine sperm.

Pairs from the same cow were in each case used for the comparison. Thedebilitated mammary glands show clear coloring at the correspondingposition at which the polyclonal antibody was applied. For cow 1, the“healthy” quarter was likewise on the way to becoming inflamed, whichindicates that the method according to the invention makes it possibleto identify mammary gland inflammation even at a very early stage. Inorder to ensure reliable discrimination between positive (that is to sayinflamed) and negative (that is to say healthy) results, a cut-off valuecan be defined, and/or reference samples can also be measured at thesame time.

1. A method for detecting inflammation in a subject comprising:obtaining a sample from bodily fluid or milk of the subject; anddetermining prostaglandin D synthase (PGDS) or parts having a length ofat least 15 amino acids in the sample, wherein determination of PGDS orparts in the sample indicates mammary gland inflammation.
 2. The methodas claimed in claim 1, wherein the subject is a human.
 3. The method asclaimed in claim 1, wherein the subject is cattle and the inflammationis mammary gland inflammation in cattle.
 4. The method as claimed inclaim 1, wherein the bodily fluid sample is chosen from plasma, synovialfluid, or urine.
 5. The method as claimed in claim 1, whereinprostaglandin D synthase (PGDS) is determined using at least oneantibody against prostaglandin D synthase (PGDS).
 6. The method asclaimed in claim 5, wherein the antibody is at least one antibodyselected from a polyclonal against prostaglandin D synthase (PGDS). 7.The method as claimed in claim 5, wherein the antibody is obtained usingas an immunogen at least one of the peptides PGDS1 (SEG ID NO:1—aminoacids 51-57 of SEQ ID NO:4), PGDS2 (SEQ ID NO:2—amino acids 6-22 of SEQID NO:4) or PGDS3 (SEQ ID NO:3—amino acids 110-125 of SEQ ID NO:4). 8.The method as claimed in claim 1, wherein prostaglandin D synthase(PGDS) is determined using immunoassay.
 9. The method as claimed inclaim 8, wherein the immunoassay is a quantitative sandwich ELISA or aWestern Blot.
 10. The method as claimed in claim 1, whereinprostaglandin D synthase (PGDS) is determined using gel electrophoresis.11. The method as claimed in claim 10, wherein the gel electrophoresisis two-dimensional gel electrophoresis.
 12. The method as claimed inclaim 1, wherein the subject is an animal.
 13. The method as claimed inclaim 5, wherein the antibody is at least one antibody selected from amonoclonal antibody against prostaglandin D synthase (PGDS).
 14. Amethod for detecting inflammation in a subject comprising: obtaining asample from bodily fluid of the subject; and determining prostaglandin Dsynthase (PGDS) or parts having a length of at least 15 amino acids inthe sample, wherein determination of PGDS or parts in the sampleindicates rheumatoid arthritis.